Process parameters influence the extracellular electron transfer mechanism in bioelectromethanogenesis

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 2 4 4 5 0 e2 4 4 5 8

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Process parameters influence the extracellular electron transfer mechanism in bioelectromethanogenesis Franziska Enzmann, Florian Mayer, Dirk Holtmann* DECHEMA Research Institute, Industrial Biotechnology, Theodor-Heuss-Allee 25, 60486, Frankfurt Am Main, Germany

highlights
Operational conditions influence the proportion of indirect electron transfer.
Ratio between direct and indirect transfer not correlated to total production rate.
Lower working electrode potential increases indirect electron transfer.
CO2 availability affects total CH4 production rate, not the electron transfer way.
Hydrogen conversion rates are usually close to 100%.

article info

abstract

Article history:

In bioelectromethanogenesis, electricity can be converted to methane, partially indirect via

Received 10 May 2019

electrochemical hydrogen production, partially via direct electron transfer to the electro-

Received in revised form

active methanogens. The electron transfer mechanism from electrode to methanogens

11 June 2019

was not fully understood so far. Using a pure culture of Methanococcus maripaludis, we show

Accepted 5 July 2019

that the ratio between direct and indirect electron transfer is shifted by altering the process

Available online 17 August 2019

conditions. The largest shift from 95.6% direct to 42.0% indirect electron transfer occurred when reducing the working potential. Further influences were observed by addition of 3-

Keywords:

(N-morpholino) propanesulfonic acid buffer and alteration of electrode material, but not by

Bioelectromethanogenesis

alteration of the in-gas composition and gas velocity. In all cases, abiotically produced

Extracellular electron transfer

hydrogen was converted at a high rate of above 75%. This research shows that the electron

Methanococcus maripaludis

uptake pathway in bioelectromethanogenesis can be influenced without genetic modifi-

Process optimization

cations, offering new possibilities for the examination of the mechanistic behind

Indirect electron transfer

electroactivity.

Hydrogen conversion rate

© 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

Introduction The use of hydrogen as fuel for vehicles is gaining increasing interest in times of global warming and depletion of fossil oil

and gas. Storage technologies such as adsorption in zeolites [1] or metal-organic frameworks [2] and capture in liquid organic hydrogen carriers [3] were suggested to increase the safety of transport, storage and use of the highly explosive gas.

* Corresponding author. E-mail address: [email protected] (D. Holtmann). https://doi.org/10.1016/j.ijhydene.2019.07.039 0360-3199/© 2019 Hydrogen Energy Publications LLC. Published by Elsevier Ltd. All rights reserved.

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Symbols and Abbreviations e I MOPS NA re,m YCH4b,EL YH2ab,EL YH2b,EL z hH2Con hind hd hC

Elementary charge 1.602*1019 As Electrical current [A] 3-(N-morpholino) propanesulfonic acid [] Avogadro constant 6.022*1023 1/mol Electron transfer rate to methane [mol/s] Specific biotic methane production rate [mol/ d*m2] Specific abiotic hydrogen production rate [mol/ d*m2] Specific biotic hydrogen production rate [mol/ d*m2] Number of electrons transferred [] Hydrogen conversion rate [%] Proportion of indirect electron transfer [%] Proportion of direct electron transfer [%] Coulombic efficiency [%]

Another possibility to store the energy contained in hydrogen would be the conversion of hydrogen to another fuel which is easier to handle [4]. A possible method could be the conversion of hydrogen to methane, which is about ten times cheaper to store than hydrogen [4]. Methane can be introduced to the natural gas grid, which is already existent in many countries [5]. The conversion of hydrogen and CO2 to methane can be realized electrochemically or biologically. The electrochemical route follows the Sabatier process, in which CO2 and H2 are converted to CH4 and water vapor [6,7]. Side reactions to alkanes or alkenes might occur, depending on the temperature and the catalyst [7]. One of the major challenges in the Sabatier process is the use of expensive and toxic catalysts like ruthenium [6] or nickel [8]. The process takes place at temperatures between 350 and 400  C [7]. The pressure can be set between 10 and 60 bar [7,8]. Biological methanation processes without the digestion of organic matter are currently designed, leading to a gas mixture containing up to 85% of methane [8]. Hydrogenotrophic methanogens are fed with CO2 and electrochemically produced H2 [8,9]. The process can be operated at up to 65  C under atmospheric pressure [8]. To our knowledge, the technology is not in a commercial state yet. Apart from the disadvantages of lower efficiency, lower production rates and lower product purity compared to the electrochemical route, the biological methanation offers advantages such as milder process conditions, increasing product specificity and lower sensitivity against substrate impurities [10]. To further intensify the biological production of methane from hydrogen and CO2, the method of bioelectromethanogenesis was developed, where hydrogen evolution by water electrolysis and biological conversion take place in the same reaction system. The conversion of CO2 to methane within bioelectromethanogenesis takes place at the cathode side of the bioelectrochemical system and is an example for a microbial electrosynthesis process [11]. The technology was described by Cheng [12] and recently reviewed [13,14]. It is supposed that the bioelectromethanogenesis allows the use of less negative potentials than needed for

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hydrogen evolution, since some electroactive methanogens seem to perform a direct electron transfer from the electrode to the microorganism for CO2 reduction, avoiding the intermediate H2 [12,15,16]. However, the complete mechanism of electron uptake was not fully understood by now. Three means of electron transfer were suggested [13]. Firstly, an abiotic electrochemical hydrogen production takes place at the cathode, which is placed in the same reactor compartment as the microorganisms. The methanogens then produce methane out of hydrogen and CO2, which is referred to as indirect electron transfer (IET) [16]. This was particularly shown for Methanothermobacter thermautotrophicus in bioelectromethanogenesis [17], but it might be valid for other methanogenic strains. An indicator therefore might be that during experiments with microbial electrolysis cells, hydrogen formation was detected during the start up phase, and methane formation started delayed together with decreasing amounts of hydrogen [18]. Hydrogenases, which activate hydrogen for further usage within the microorganisms, would be involved in the indirect bioelectromethanogenesis by taking up hydrogen [19]. Some hydrogenases are reversible and can also convert Hþ and electrons to H2 in a bio-catalyzed hydrogen formation [20]. It was suggested that this mechanism is also used in several electroactive bacteria to store excess electrons [21]. The additionally bio-produced hydrogen could also be used for bioelectromethanogenesis in a kind of self-induced indirect electron transfer. This mechanism is not distinguishable from direct electron transfer by comparing abiotical and biotical gas production rates. It was shown that electron uptake without hydrogenases is also possible [22]. The methanogens accept the electrons directly from the cathode surface and produce methane using electrons, protons and CO2, which allows less negative working potentials at the cathode than needed for hydrogen evolution. This direct electron transfer (DET) could happen via surface proteins (e.g. cytochromes) or conductive filaments [12]. It is possible that cytochromes may play a role in direct electron uptake from an electrode in cytochromecontaining methanogens, as for example Methanosarcina [23]. Since not all methanogens, which are considered electroactive by direct electron transfer, do contain cytochromes, other means of DET must be involved. As an example, hydrogenases, which are present in all wild type methanogenic strains, are considered to be important for the direct electron uptake, too [24]. In this publication, a wild type of the electroactive methanogen Methanococcus maripaludis was used. This microorganism was already described for the use in bioelectromethanogenesis [22] and several enzymes like hydrogenases and formate dehydrogenases were suggested to participate in the extracellular electron transfer [25]. These enzymes seem to be released by the cells and attach to the cathode surface, allowing a kind of selfinduced indirect electron transfer via hydrogen and formate [25]. An electron bifurcuration pathway could also be essential for extracellular electron uptake [26]. The electrons could also be transferred to the methanogens via mediator molecules in a mediated electron transfer, although this was not shown for methanogens so far [27]. It is not yet known, which methanogens are electroactive at all, not to mention which electron uptake mechanisms they perform in detail, but it is supposed that all means of electron uptake contribute to the overall process [28]. Since the

RVC foam

The electrochemical bubble column reactor was described before [30]. The working chamber of the two chamber system was designed as bubble column holding 1 l working volume (MES medium, described in [30]) with a surrounding counter chamber holding 10 l of 0.1 M phosphate buffer (pH 6.8). Different working electrodes were applied in the center of the working chamber; the counter electrode was carbon fabric (0.06 m2 geometrical surface area, ACC5092-15, Kynol, Hamburg, Germany). The membrane windows were covered with proton exchange membrane (0.012 m2, Nafion 117, DuPont, Welmington, USA).

Carbon, 45 ppi (pores per inch) ERG Aerospace Corp., Oakland, USA 0.0124 4.196

Reactor setup

Graphite Metallpulver 24, St. Augustin, Germany 0.0069 25.231 Material Supplier Geometrical surface area [m2] Specific surface area [m2/g] (measured by physisorptiona)

Graphite rod

For the different comparison experiments, different operational conditions were applied. To observe the effect of applied working potential, a graphite rod (0.0069 m2 geometrical surface area) was used as working electrode and a potential of 0.9 V and 1.1 V vs Ag/AgCl were applied, respectively, using a reference electrode (Ag/AgCl electrode; þ199 mV vs. SHE, SE 21, Sensortechnik Meinsberg, Xylem Analytics, Germany) placed in a luggin capillary (Fischer Labortechnik, Frankfurt am Main, Germany) filled with 0.5 M Na2SO4) and connected to a potentiostat (Multimaster 2.1, Materials Mates, Milano, Italy). As in-gas, N2/CO2 80/20 (v/v) was used at a gassing rate of 30 ml/min (0.03 vvm). The effect of different working electrode materials was tested at an applied potential of 1.1 V vs Ag/AgCl using four electrode materials as given in Table 1. The gassing conditions were as for the potential comparison. The comparison of different gassing conditions was done with 2 graphite rods (0.0138 m2) as cathode poised at 1.1 V vs Ag/AgCl. Different in-gas composition (N2/CO2 in 80/20, 50/50 and 0/100 v/v, respectively) were used at 30 ml/min. Different in-gas velocities of 30 ml/min, 60 ml/min and 90 ml/min, respectively, were tested with pure CO2 as in-gas. Using a larger RVC foam (0.0248 m2) poised at 1.1 V vs Ag/AgCl under

Table 1 e Different electrodes materials used as cathode.

Operation

The physisorption measurement is a standard measuring technology for solid surfaces based on nitrogen adsorption on the material [31]. For evaluation, a BET isotherm (after Brunauer, Emmet and Teller) was used; The values given here are shown to give an impression of the large differences of the surface structure of these carbon based materials, specific production rates are always calculated based on the geometrical surface area.

Materials and methods

a

Carbon fabric

Carbon laying

electron uptake is a surface dependent mechanism in any case, the design of a bioelectrochemical reactor and the integration of a suitable electrode are crucial for the efficiency of the process, among other operational conditions of bioelectromethanogenesis [29]. In this publication, we show that the extracellular electron transfer from electrode to the electroactive methanogen Methanococcus maripaludis is dependent on the process conditions, leading to different proportions of direct and indirect electron transfer. We always refer to “direct” electron transfer, if a direct biological interaction with the electrode is needed, this comprises the conventional direct electron transfer and the “self-induced indirect” electron transfer via biotically produced hydrogen. The indirect electron transfer in this paper is the indirect transfer via abiotically produced hydrogen. Furthermore, we show the hydrogen conversion rates under different process conditions.

Carbon, HP-T450C HP textiles, Schapen, Germany 0.0057 0.888

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Activated carbon, ACC5092-15 Kynol, Hamburg, Germany 0.0041 1635.293

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a gas flux of 30 ml/min N2/CO2 80/20 (v/v), the addition of 20.9 g/l MOPS buffer to the MES medium was tested.

Mediated electron transfer was not considered since no mediators were added to the medium.

Preculture

hind ¼ 100 % 

The experiments were done in two biologically independent replicates and one abiotic control for 90 h each. All chronoamperometric experiments were conducted at 35  C. Methanococcus maripaludis S2 (DSM No.: 14266, DSMZ, Braunschweig, Germany) was used as electroactive microorganism. Pre-cultures were cultivated at 180 rpm and 37  C in 1 l septum flasks with 300 ml of M141 medium under H2/CO2 (80/20, v/v) gas atmosphere pressurized to 2 bar. The optical density of the pre-cultures used for inoculation was approximately 1. The working chambers of the reactors were inoculated to an OD600 of 0.1 after anaerobization for 1 h and potential was applied at the same time, so no acclimation period for the cells was used.

Analytics and calculations Gas samples were taken from the reactors daily and analyzed via gas chromatography (Agilent technologies 490 Micro GC, Agilent, Santa Clara, USA (with external 2-point-calibration)). For analysis of the off gas samples, an injector temperature of 100  C and a column temperature of 60  C were set. Samples were injected to three columns: Channel 1: PoraPLOT U precolumn and Molsieve 5A main column with argon as carrier gas; Channel 2 PoraPLOT U pre-column and Molsieve 5A main column with helium as carrier gas; Channel 3 PoraPLOT U as pre-column and main column with helium as carrier gas. A thermal conductivity detector was used. Hydrogen was detected on channel 1, oxygen and nitrogen on channel 2 and methane and carbon dioxide on channel 3. The sampling time was set to 30 s, the total runtime to 3 min. The pH (VoltcraftPH100ATC; Voltcraft, Hirschau, Germany) and conductivity (HI99301 conductivity meter, Hanna instruments, € hringen, Germany) in both chambers were measured daily. Vo The optical density in the working was also measured daily (WPA Biowave CO8000 Cell Density Meter, 600 nm, Biochrom Ltd, Cambridge, Great Britain). The current was monitored continuously. The hydrogen conversion rate was calculated using the abiotic hydrogen production and the residual hydrogen detected in biotic experiments (Equation (1)); the biotically catalysed hydrogen production was not considered because it was assumed that biotically produced hydrogen was metabolized straight away by the methanogens. hH2Con ¼ 100 % 

YH2ab;EL  YH2b;EL YH2ab;EL

(1)

The proportion if indirect methane production via abiotically produced hydrogen was calculated using the abiotic hydrogen production, the residual hydrogen detected in biotic experiments and the biotic methane production (Equation (2), Equation (3) for proportion of direct electron transfer). It cannot be distinguished between direct and self-induced indirect extracellular electron transfer, so these two ways were summarized as direct electron transfer since they both require a direct biological interaction with the cathode.

YH2ab;EL  YH2b;EL 4  YCH4b;EL

hd ¼ 100 %  hind

(2)

(3)

The Coulombic efficiency was calculated using equation (4); therefore, the mean values of production rates and current were used, leading to one mean Coulombic efficiency for each experiment. hC ¼ e  NA

z  re;m I

(4)

Results During all experiments, a current consumption by the methanogens was observed and stabilized after a polarization time of approximately 20 h. Therefore, all mean values were calculated from 24 h after the inoculation until the end of the experiment to exclude any effects from the electrode polarization and the inoculation. Changes of the optical density in the biotic experiments were not detected, leading to the conclusion that no growth of the methanogens took place; biofilms at the electrodes were not observed. In all biotic experiments, methane could be detected via GC analysis. In the abiotic runs, no methane was observed. After the polarization time, the current density in the biotic runs remained relatively constant, as well as the methane and biotic hydrogen production rate. An example is given in Fig. 1.

Abiotic hydrogen production In all abiotic measurements, hydrogen production was observed, although the specific hydrogen production rates varied from 4.7 mmol/d*m2 and 652.9 mmol/d*m2 between the different conditions (Table 2). The variation of the potential from 0.9 V vs Ag/AgCl to 1.1 V vs Ag/AgCl resulted in an increase of the abiotic hydrogen production rate by the factor of 21.5 with an improved Coulombic efficiency for abiotic hydrogen production from 10.5 to 29.2 (Table 2, entries 8 and 9)). The abiotic hydrogen production rates for the graphite rod, the RVC foam and the carbon laying cathode were similar between 144.1 and 225.3 mmol/d*m3 with the highest Coulombic efficiency for abiotic hydrogen production with the carbon laying (41.4%, Table 2, entry 12), whereas the carbon fabric cathode produced significantly less hydrogen (26.3 mmol/d*m2) at a very low Coulombic efficiency of only 5.4% (Table 2, entry 11). The electron flux not explainable by hydrogen evolution probably resulted in alterations of the surface charges of the electrode. Carbon fabric offers a high specific surface area, which could lead to the small amount of electrons transferred to hydrogen. An increased CO2 content from 20% to 100% in the in-gas also enhanced the abiotic hydrogen production by a factor of 2.1. The increase of the gassing rate from 30 ml/min to 90 ml/ min did increase the abiotic hydrogen production rate by 14%, but considering the deviation of the results this was not

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20

40

60

80

100

0 -1

-2 -3 -4 -5 -6

B

4

2 1.8 1.6 1.4 1.2 1 0.8 0.6 0.4 0.2 0

3.5 3 2.5 2

1.5 1 0.5

CH4 produc on [ml/h]

Curre nt de nsitiy [A/m²]

0

H2 produc on [ml/h]

Time [h]

A

0 0

20

40

60

80

100

Time [h]

Fig. 1 e Current consumption and gas production with M. maripaludis. Performance of a biotic run with two graphite rods as working electrode (0.0138 m2 geometrical surface area), pure CO2 as in-gas (90 ml/min) and an applied potential of ¡1.1 V. Mean values out of two biologically independent runs with mean deviation. A) Current density B) Dots: Methane production rate; Crosses: biotic hydrogen production rate.

significant (Table 2, entries 3, 4 and 5). The additional hydrogen production when using higher CO2 contents in the in-gas was likely to result from the lowering of the pH of the medium from 7.9 to 7.1 due to the larger CO2 concentration. This improved the proton availability and therefore allowed higher hydrogen evolution rates. The addition of MOPS buffer caused an increase of the abiotic hydrogen production rate by a factor of 12.7. It was already shown before that MOPS can act as electron donor or electron shuttle [32], thus it might be possible that it also catalysed the abiotic hydrogen production.

additionally produced hydrogen effectively. The conversion rate improves again from 76.3% to 85.6% when increasing the in-gas velocity, probably due to better mixing conditions and higher CO2 availability for the microorganisms. MOPS buffer also slightly improved the hydrogen conversion rate by 9.8%, maybe by stabilizing enzymes responsible for hydrogen uptake [32]. The high conversion rates suggest that bioelectromethanogenesis is an effective way to convert hydrogen to methane without hydrogen losses. The storage of energy in methane instead of in hydrogen leads to improved storage and transportation properties.

Hydrogen conversion

Methane production and ratio between direct and indirect electron transfer

In most experiments, little residual H2 was detected in the offgas of the biotic runs (highest 135.1 mmol/d*m2, 28.7 mmol/ d*m2 in average); only when using the RVC foam electrode at a working potential of 1.1 V vs Ag/AgCl, 100% of the abiotically produced hydrogen were converted to methane (Table 2, entry 10). In all other experiments, the conversion rate was lower, but always above 75%, even if the abiotic production rates was above 500 mmol/d*m2 (Table 2). The solubility of hydrogen in water is low under atmospheric pressure between 0  C and 66  C [33]; it might be that the transport of the abiotically produced hydrogen out of the medium was slightly faster than the conversion by the microorganisms. The macro-porous structure of the RVC foam could capture the gas bubbles, thus increasing the retention time of hydrogen in the medium, allowing a full conversion. The lowest conversion rate of 75% was calculated using a carbon fabric cathode at 1.1 V vs Ag/AgCl. The methane production and the abiotic hydrogen production under this condition were in general lower (3.4e4.0 times decreased methane production, 5.5 to 8.6 times decreased abiotic hydrogen production) than for other used electrodes; the carbon fabric seemed to hinder the hydrogen evolution as well as the effective interaction of the methanogens with the electrode surface. The conversion rate also decreased from 97.3% to 76.3% when altering the CO2 content in the in-gas from 20% to 100%. In contrast, the total amount of converted hydrogen was doubled since the abiotic hydrogen production increased; thus, it is possible that the metabolism of the methanogens was too slow to convert the

Observing the specific production rates of methane for the different process conditions, it is clear that gassing as well as electrical conditions had an impact on the process performance and overall methane production (Table 2). The methane production rate varied between 20.5 mmol/d*m2 (entry 11) and 195.3 mmol/d*m2 (entry 3). The decreased working potential of 1.1 V vs Ag/AgCl improved mainly the indirect electron transfer from 1.5 mmol/d*m2 to 34.5 mmol/d*m2, but less the direct electron transfer, which only increased from 32.4 mmol/d*m2 to 47.8 mmol/d*m2. The overall methane production rate increased by a factor of 2.4 when decreasing the potential from 0.9 V to 1.1 V vs Ag/AgCl. The amount of methane explainable by direct electron transfer was only affected by a factor of 1.4, the amount of methane via indirect electron transfer, in contrast, increased by a factor of 23.4. The ratio of direct to indirect electron transfer was therefore shifted towards indirect electron transfer (Fig. 2). A shift from direct to indirect electron transfer at decreased potentials was already suggested before and could be confirmed in this study [16,34]. The Coulombic efficiency of methane production slightly decreased from 51.0% to 47.3%, although the efficiency of abiotic hydrogen production was increased by a factor of 2.8 at the lower potential and (Table 2, entry 9). The graphite rod electrode with a direct methane production of 47.8 mmol/ d*m2 seemed to allow a better direct electron transfer between microorganism and electrode surface than the three other

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97.3 96.3 76.3 88.2 85.6 87.3 97.1 88.3 95.8 100.0 75.0 97.2 42.9 43.2 29.9 31.5 40.5 15.9 32.9 10.5 29.2 35.3 5.4 41.1

GR, -0.9 V

267.1 ± 11.6 341.9 ± 71.7 569.3 ± 53.9 619.6 ± 109.0 652.9 ± 247.6 4.7 ± 1.2 59.9 ± 14.7 6.7 ± 1.3 144.1 ± 30.8 202.8 ± 18.6 26.3 ± 9.5 225.3 ± 27.7 7.2 ± 3.3 12.8 ± 6.0 135.1 ± 36.7 73.4 ± 8.5 94.2 ± 44.3 0.6 ± 0.5 1.7 ± 2.3 0.8 ± 0.6 6.0 ± 2.4 0.0 ± 0.0 6.6 ± 4.1 6.3 ± 2.0 20% CO2, 30 ml/min 50% CO2, 30 ml/min 100% CO2, 30 ml/min 100% CO2, 60 ml/min 100% CO2, 90 ml/min 20% CO2, 30 ml/min 20% CO2, 30 ml/min 20% CO2, 30 ml/min 20% CO2, 30 ml/min 20% CO2, 30 ml/min 20% CO2, 30 ml/min 20% CO2, 30 ml/min 1 2 3 4 5 6 7 8 9 10 11 12

Graphite rod, 0.0138 m2 Graphite rod, 0.0138 m2 Graphite rod, 0.0138 m2 Graphite rod, 0.0138 m2 Graphite rod, 0.0138 m2 RVC foam, 0.0248 m2 (w/o MOPS) RVC foam, 0.0248 m2 (w/MOPS) Graphite rod, 0.0069 m2 Graphite rod, 0.0069 m2 RVC foam, 0.0124 m2 Carbon fabric, 0.0041 m2 Carbon laying, 0.0057 m2

1.1 1.1 1.1 1.1 1.1 1.1 1.1 0.9 1.1 1.1 1.1 1.1

78.1 ± 40.4 89.29 ± 15.1 143.7 ± 33.8 189.9 ± 11.8 195.3 ± 12.7 36.0 ± 5.6 37.6 ± 14.7 33.8 ± 1.7 82.3 ± 10.7 70.6 ± 1.2 20.5 ± 9.2 76.4 ± 11.3

46.7 44.1 60.1 70.4 75.6 56.4 41.4 51 47.3 55.6 27.4 45.6

Abiotic H2 production [mmol/d*m2]

0%

Biotic H2 production [mmol/d*m2] Coulombic efficiency (biotic CH4) [%] Biotic CH4 production [mmol/d*m2]

CF, 1.1 V

GR, -1.1 V

Gas composition and gassing rate

Working potential [V vs Ag/AgCl]

CL, -1.1 V

RVC, -1.1 V

Electrode type and geometrical surface area Entry

Table 2 e Performance of bioelectromethanogenesis under different operational conditions.

Coulombic efficiency (abiotic H2) [%]

H2 conversion rate [%]

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20%

40%

60%

80%

100%

Electron transfer Fig. 2 e Ratio between direct and indirect electron transfer under different electrical conditions. GR: graphite rod cathode, RVC: reticulated vitreous carbon cathode, CF: carbon fabric; CL: carbon laying; Potentials given vs Ag/ AgCl; Dark grey: proportion of indirect electron transfer; Light grey: proportion of direct electron transfer, calculated from mean values of specific methane and hydrogen production rates in biotic and abiotic experiments.

electrodes, which show similar amounts of methane produced by direct electron transfer between 15.5 mmol/d*m2 and 21.7 mmol/d*m2. RVC foam and carbon laying, which allowed hydrogen production at a slightly higher Coulombic efficiency above 35%, shifted the methane production towards more than 50 mmol/d*m2 indirect electron transfer. With the carbon fabric electrode, hydrogen could not be produced efficiently, so that only 4.9 mmol/d*m2 methane explainable via indirect electron transfer was detected (Table 2, entry 11 and Fig. 2). An increased CO2 availability increased the overall methane production from 78.1 mmol/d*m2 at 20% CO2 in the in gas at 30 ml/min gas flux to 195.3 mmol/d*m2 at 90 ml/min pure CO2 gas flux (Table 2, entries 1 to 5). Both, direct and indirect electron transfer seemed to benefit from the increased CO2 availability. The ratio between direct and indirect electron transfer was not significantly affected by the gassing conditions (Fig. 3). The indirect electron transfer outcompeted the direct electron transfer and was responsible for around 70% of the methane produced, which corresponds with the higher abiotic hydrogen production rates obtained for higher CO2 contents and gassing rates (Table 2). Also, the Coulombic efficiency for methane production increased from 60.1% to 75.6% with increased in-gas velocity (Table 2, entries 3, 4 and 5). Two possible reasons were suggested. On the one hand, the improved mixing conditions could enhance the contact frequency of the methanogens with the electrode, increasing the direct electron transfer. On the other hand, the improved substrate availability overcame carbon limitations during the process, resulting in a more efficient conversion of the electrical current. The addition of MOPS buffer enhanced the indirect electron transfer from 31.6 mmol/d*m2 to 108.6 mmol/d*m2, but not the direct electron transfer, which decreased from 102.3 to 35.1 mmol/d*m2. The Coulombic efficiency of methane production also decreased by 15% (Table 2, entries 6 and 7 and

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value in terms of energy conversion. No correlation was observed between the ratio of direct to indirect electron transfer and the Coulombic efficiency. Thus, it cannot be concluded whether the direct or the indirect electron transfer is more efficient in terms of Coulombic efficiency.

gr3, 100 %

gr2, 100 % gr1, 100%

Discussion gr1, 50% gr1, 20 % 0%

20%

40%

60%

80% 100%

Electron transfer Fig. 3 e Ratio of direct and indirect electron transfer under different gassing conditions. gr1: gassing rate 1, 30 ml/min; gr2: gassing rate 2, 60 ml/min; gr3: gassing rate 3, 90 ml/ min; percentages: CO2 content in the in-gas stream; Dark grey: proportion of indirect electron transfer, Light grey: proportion of direct electron transfer, calculated from mean values of specific methane and hydrogen production rates in biotic and abiotic experiments.

Fig. 4). This suggested that the by a factor of 12.7 increased abiotic hydrogen production alone was responsible for the slightly improved total methane production, not an effect of the MOPS buffer on the enzyme stability or the microorganisms. This also showed that MOPS did not act as an electron shuttle between electrode and microorganism, which would not be distinguishable from a direct electron transfer in the experimental setup and therefore increase the methane portion by direct electron transfer. The ratio between direct and indirect electron transfer was thus shifted towards indirect electron transfer [Fig. 4). The Coulombic efficiency of methane production was in all cases slightly higher than for the abiotic hydrogen production (Table 2), confirming that the use of a biocatalyst can add

w/ MOPS

w/o MOPS

0%

20%

40%

60%

80%

100%

Electron transfer Fig. 4 e Shifted ratio between direct and indirect electron transfer by the addition of MOPS buffer. Dark grey: proportion of indirect electron transfer; Light grey: proportion of direct electron transfer, calculated from mean values of specific methane and hydrogen production rates in biotic and abiotic experiments.

Based on the results, it is obvious that many process parameters influence bioelectromethanogenesis not only in terms of production rates and efficiency, but also regarding the mechanistic interactions between electrode and microorganism. To further elucidate the pathways of extracellular electron transfer, real-time PCR and proteomics could be used to detect up-regulations in the genome. This could then clarify which enzymes take part in the electron uptake and which molecular changes improve the electron uptake. Knowing the exact function of the increased or suppressed metabolic activities, it might be possible to finally explain the extracellular transfer pathway in methanogens. This can in further development serve as a starting point for the genetic modification of electroactive microorganisms, allowing higher productivities and efficiencies. Increasing knowledge of the extracellular electron transfer pathways could also speed up the development of completely new biocatalysts for the conversion of electricity to more valuable compounds by genetic engineering. This would be a further step towards industrial feasibility of the microbial electrosynthesis technology. The technology of bioelectromethanogenesis aims to contribute to the replacement of fossil energy and chemical resources. Therefore, it is necessary to further optimize the process to increase the Coulombic and energy efficiency, the CO2 fixation rate and the methane production rate. According to the results, optimization potential can especially be found in the development of low-resistance electrode materials, allowing a better direct electron transfer to the microorganisms. Other process parameters to optimize would be the reactor-design in terms of resistance reduction and materials for counter electrode and membrane. An evaluation of the maturity of bioelectromethanogenesis is given by the technology readiness level (TRL) [35]. The TRL shows, how far a technology or process already proceeded from the research idea (TRL 1) to industrial application (TRL 9). Based on the current research, we suggest to place bioelectromethanogenesis in TRL 6, which says that a prototype operation in a relevant environment was demonstrated, as it was shown for first pilot plant reactors [36] and during first long-term experiments [34]. A higher level cannot be recommended, since experiments under real industrial conditions like failure simulations or fluctuation tests were not shown. For most bioelectrochemical synthesis processes, the TRL is lower [37]. It has to be mentioned, that the operation of a pilot plant and thereby the suggested TRL is only valid for mixed culture experiments with additional organic substrate, not for pure culture investigations with CO2 as sole carbon source; for the technology shown here with a pure culture, a TRL of 4e5 would be more adequate. Scalability studies of bioelectromethanogenesis with pure cultures was not reported so far, although first suggestions for scale up parameters were

i n t e r n a t i o n a l j o u r n a l o f h y d r o g e n e n e r g y 4 4 ( 2 0 1 9 ) 2 4 4 5 0 e2 4 4 5 8

made [29,30]. A main challenge in future will be to turn bioelectromethanogenesis into a feasible technology for methane production compared to the well established Sabatier process [6] and biomethanation of CO2 and hydrogen, which is on the turn to industrial application [38,39]. It has to be competitive to these technologies in terms of energy efficiency, costs and CO2 footprint. By now, the data basis is not yet sufficient to predict if this goal will be reached.

Conclusion By now, the complete mechanism of extracellular electron transfer between an electroactive microorganism and an electrode was not completely elucidated. Two main electron transfer mechanisms were separated in this paper, the direct electron transfer in which the microorganism or enzymes directly interact with the electrode, and the indirect electron transfer, whereby microorganisms convert abiotically produced hydrogen to methane. We could show, as it was suggested before, that both mechanisms contribute to a methane production with M. maripaludis in a bioelectrochemical system, but the ratio of direct to indirect electron transfer can be shifted using different process conditions. It was shown previously that the applied potential can influence the ratio between direct and indirect electron transfer, but many more factors obviously affect the mechanism. So surprisingly, the electron transfer pathway is not only dependent on the electroactive strain and the working potential used, but also on other operational parameters. The strongest influence on the ratio between direct and indirect electron transfer was observed by the addition of redox-active medium compounds and alteration of the working potential. Interestingly, also different carbonbased materials show different proportions of indirect electron transfer, in accordance with their hydrogen evolution rate, which was not observed in previous studies. On the contrary, alterations of the gassing conditions could improve the overall methane production rate without affecting the ratio between direct and indirect electron transfer. A correlation between Coulombic efficiency and proportion of direct electron transfer rate could not be observed, which shows that the efficiency of both electron transfer pathways can be similar; in contrast, it was suggested before that direct electron transfer might result in a more efficient electron recovery and should therefore be preferred [40]. In all experiments, abiotic hydrogen was converted to methane very effectively at higher Coulombic efficiencies than calculated for abiotic hydrogen production, suggesting that bioelectromethanogenesis can be a suitable and selective technology for the storage of electrical energy.

Acknowledgements The authors thank the BMBF for funding the project “MIKE e Methanation of CO2 from biogas by microbial electrosynthesis (FKZ: 033RC013A) and all project partners for fruitful discussions.

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